The present disclosure relates generally to methods for isolating molecules from a fluidic sample. More particularly, the present disclosure relates to methods for isolating weakly interacting molecules from a complex mixture using an immiscible phase filtration technique.
The isolation of proteins, nucleic acids, and small molecules from a complex biological mixture, such as a cell lysate or whole blood, is important in a broad array of fields, including biology, diagnostics, biochemistry, pharmacology, and forensics. In particular, molecule-molecule interactions such as, for example, protein-protein interactions, nucleic acid-nucleic acid interactions, protein-nucleic acid interactions, protein-small molecule interactions, and nucleic acid-small molecule interactions, among others, are important in a wide variety of cellular events. Because of the significance of molecule-molecule interactions, a number of physical, molecular biological, and genetic methods have been developed to isolate and identify molecular interactions.
Protein affinity chromatography, for example, uses a protein coupled to a matrix to isolate proteins that interact with the matrix-coupled protein. In this technique, non-interacting proteins are readily washed away under low-salt conditions, while the interacting proteins are retained on the matrix. Immunoprecipitation (IP) is another isolation technique that uses antibody (Ab)-bound scaffolds such as, for example, agarose beads or paramagnetic particles (PMPs), to selectively bind a protein of interest. As with affinity chromatography, after the antibody binds its antigen, a series of washing steps are performed to remove unbound protein, nucleic acids, and cell debris, as well as residual lysis buffer, which may impede downstream analyses such as mass spectroscopy (MS).
Multi-step solid phase extraction may be used to isolate and purify nucleic acids. Solid phase extraction involves binding nucleic acids to an immobilized solid phase. Bound nucleic acids are repeatedly washed to remove contaminants before they are eluted for downstream processing. High throughput versions of solid phase extraction process based on microtiter plate architectures are also commercially available, but these processes are labor intensive and can require expensive robotics to facilitate the extensive washing that must be performed on individual samples, which again limits widespread adoption of these techniques.
Recently, researchers have developed microfluidic embodiments of immunoprecipitation. However, such techniques typically require multiple washing steps which increase the complexity of microfluidic device design and operation, thus hindering the implementation of such platforms within non-engineering disciplines. Alternative techniques have been developed that rely on principles such as nano-sieving or partitioning into PEG-rich fluids using genetically-engineered tags, but these techniques possess a fair degree of microfluidic engineering complexity. While microfluidic embodiments can provide practical advantages (e.g. reduced reagent consumption, increased automation, lower device cost, and enhanced throughput), they have not offered significant improvements in the isolation of proteins or other molecules.
While the methods described above are suitable for isolating strongly interacting molecules, the required washing steps in these methods can inadvertently wash away weakly interacting molecules. Consequently, the isolation of weakly interacting molecules has been technically challenging. Accordingly, there exists a need to develop methods for isolating the wide variety of biologically important, but weakly interacting molecules.